Modified Cell with Enhanced Migration Capability

ABSTRACT

Embodiments relate to a modified T cell comprising an antigen binding molecule, wherein expression and/or function of CDC42 in the modified cell has been enhanced. In embodiments, the modified cell has an increased level of cytokine release in response to an antigen that the antigen binding molecule binds as compared to a corresponding T cell that does not overexpress CDC42. In embodiments, the cytokine release comprises a cytokine release of IFNγ. In embodiments, the modified cell has an enhanced migration capability in response to a chemokine as compared to a corresponding T cell that does not overexpress CDC42.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/775,168, filed Dec. 4, 2018, which are incorporated by referenceherein in its entirety.

SEQUENCE LISTING INFORMATION

A computer readable textfile, entitled “Sequence Listing_ST25.txt,”created on or about Nov. 12, 2019, with a file size of about 61 KB,contains the sequence listing for this application and is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to compositions and methods related tochimeric antigen receptor cells and uses thereof in the treatment ofdiseases, including cancer.

BACKGROUND

Cancer is known as malignant tumors involving abnormal cell growth withthe potential to invade or spread to other parts of the body. In humans,there are more than one hundred types of cancer. Normally, once cancercells are exfoliated, they spread over the entire body via the bloodand/or lymph systems and therefore become life-threatening. Currently,CAR-T therapy appears not to be effective for treating solid tumorcancers. One of the challenges is that CAR-T cells seem not to be ableto migrate effectively towards these cancer cells spreading over thebody.

SUMMARY

Embodiments relate to a modified T cell comprising an antigen bindingmolecule, wherein expression and/or function of CDC42 in the modifiedcell has been enhanced. In embodiments, the modified cell has anincreased level of cytokine release in response to an antigen that theantigen binding molecule binds as compared to a corresponding T cellthat does not overexpress CDC42. In embodiments, the cytokine releasecomprises a cytokine release of IFNγ or GRAM B. In embodiments, themodified cell has an enhanced migration capability in response to achemokine as compared to a corresponding T cell that does notoverexpress CDC42.

This Summary is not intended to identify key features or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The Detailed Description is described with reference to the accompanyingfigures. The use of the same reference numbers in different figuresindicates similar or identical items.

FIG. 1 shows an example of CAR construct and examples of CAR T cells.

FIG. 2 shows cytometry assay results indicating that CDC42 sequence doesnot affect the expression of CAR on T cells.

FIG. 3 shows cytometry assay results indicating that CDC42 sequence doesnot affect the activation of CAR T cells.

FIG. 4 shows CDC42 expression enhances the release of Gram B and IFN-γafter CAR activation.

FIG. 5 shows that CDC42 was successfully expressed in CAR T cells.

FIG. 6 shows that the migration ability of GUCY2C CAR T cells expressingCDC42 was significantly stronger than that of the control group.

FIG. 7 shows cytometry assay results indicating that ShRNA1 sequencedoes not affect the expression of CAR on T cells and also showscytometry assay results indicating that ShRNA1 sequence does not affectthe activation of CAR T cells.

FIG. 8 shows ShRNA1 expression enhances the release of Gram B and IFN-γ.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any method andmaterial similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, preferred methods andmaterials are described. For the purposes of the present disclosure, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “activation,” as used herein, refers to the state of a cellthat has been sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody” is used in the broadest sense and refers tomonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multi-specific antibodies (e.g., bispecificantibodies), and antibody fragments so long as they exhibit the desiredbiological activity or function. The antibodies in the presentdisclosure may exist in a variety of forms including, for example,polyclonal antibodies; monoclonal antibodies; Fv, Fab, Fab′, and F(ab)₂and fragments; as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antibody fragments” refers to a portion of a full-lengthantibody, for example, the antigen binding or variable region of theantibody. Other examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multi-specific antibodies formed from antibodyfragments.

The term “Fv” refers to the minimum antibody fragment which contains acomplete antigen-recognition and -binding site. This fragment consistsof a dimer of one heavy- and one light-chain variable region domain intight, non-covalent association. From the folding of these two domainsemanates six hypervariable loops (3 loops each from the H and L chain)that contribute amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv including only three complementaritydetermining regions (CDRs) specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site (the dimer).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. An “antibody light chain,” asused herein, refers to the smaller of the two types of polypeptidechains present in all antibody molecules in their naturally occurringconformations. K and A light chains refer to the two major antibodylight chain isotypes.

The term “synthetic antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage. The term also includes an antibody whichhas been generated by the synthesis of a DNA molecule encoding theantibody and the expression of the DNA molecule to obtain the antibodyor to obtain an amino acid encoding the antibody. The synthetic DNA isobtained using technology that is available and well known in the art.

The term “antigen” refers to a molecule that provokes an immuneresponse, which may involve either antibody production, or theactivation of specific immunologically-competent cells, or both.Antigens include any macromolecule, including all proteins or peptides,or molecules derived from recombinant or genomic DNA. For example, DNAincluding a nucleotide sequence or a partial nucleotide sequenceencoding a protein or peptide that elicits an immune response, andtherefore, encodes an “antigen” as the term is used herein. An antigenneed not be encoded solely by a full-length nucleotide sequence of agene. An antigen can be generated, synthesized or derived from abiological sample including a tissue sample, a tumor sample, a cell, ora biological fluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect associated with a decrease in tumor volume, a decrease in thenumber of tumor cells, a decrease in the number of metastases, decreasein tumor cell proliferation, decrease in tumor cell survival, anincrease in life expectancy of a subject having tumor cells, oramelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells, and antibodies inthe prevention of the occurrence of tumors in the first place.

The term “auto-antigen” refers to an endogenous antigen mistakenlyrecognized by the immune system as being foreign. Auto-antigens includecellular proteins, phosphoproteins, cellular surface proteins, cellularlipids, nucleic acids, glycoproteins, including cell surface receptors.

The term “autologous” is used to describe a material derived from asubject that is subsequently re-introduced into the same subject.

The term “allogeneic” is used to describe a graft derived from adifferent subject of the same species. As an example, a donor subjectmay be a related or unrelated or recipient subject, but the donorsubject has immune system markers that are similar to the recipientsubject.

The term “xenogeneic” is used to describe a graft derived from a subjectof a different species. As an example, the donor subject is from adifferent species than a recipient subject, and the donor subject andthe recipient subject can be genetically and immunologicallyincompatible.

The term “cancer” is used to refer to a disease characterized by therapid and uncontrolled growth of aberrant cells. Cancer cells can spreadlocally or through the bloodstream and lymphatic system to other partsof the body. Examples of various cancers include breast cancer, prostatecancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma,leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “includes” and “including” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

The phrase “consisting of” is meant to include, and is limited to,whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory and that no other elements may be present.

The phrase “consisting essentially of” is meant to include any elementlisted after the phrase and can include other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base-pairing rules, or theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

The term “corresponds to” or “corresponding to” refers to (a) apolynucleotide having a nucleotide sequence that is substantiallyidentical or complementary to all or a portion of a referencepolynucleotide sequence or encoding an amino acid sequence identical toan amino acid sequence in a peptide or protein; or (b) a peptide orpolypeptide having an amino acid sequence that is substantiallyidentical to a sequence of amino acids in a reference peptide orprotein.

The term “co-stimulatory ligand,” refers to a molecule on anantigen-presenting cell (e.g., an APC, dendritic cell, B cell, and thelike) that specifically binds a cognate co-stimulatory molecule on a Tcell, thereby providing a signal which, in addition to the primarysignal provided by, for instance, binding of a TCR/CD3 complex with anMHC molecule loaded with peptide, mediates a T cell response, includingat least one of proliferation, activation, differentiation, and othercellular responses. A co-stimulatory ligand can include B7-1 (CD80),B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatoryligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40,CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6,ILT3, ILT4, HVEM, a ligand for CD7, an agonist or antibody that bindsthe Toll ligand receptor and a ligand that specifically binds withB7-H3. A co-stimulatory ligand also includes, inter alia, an agonist oran antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds CD83.

The term “co-stimulatory molecule” refers to the cognate binding partneron a T cell that specifically binds with a co-stimulatory ligand,thereby mediating a co-stimulatory response by the T cell, such asproliferation. Co-stimulatory molecules include an MHC class I molecule,BTLA, and a Toll-like receptor.

The term “co-stimulatory signal” refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules. The terms “disease” and “condition” may be usedinterchangeably or may be different in that the particular malady orcondition may not have a known causative agent (so that etiology has notyet been worked out), and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians. Theterm “disease” is a state of health of a subject wherein the subjectcannot maintain homeostasis, and wherein if the disease is notameliorated, then the subject's health continues to deteriorate. Incontrast, a “disorder” in a subject is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

The term “effective” refers to adequate to accomplish a desired,expected, or intended result. For example, an “effective amount” in thecontext of treatment may be an amount of a compound sufficient toproduce a therapeutic or prophylactic benefit.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as a template for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene encodes a protein if transcription and translation of mRNAcorresponding to that gene produces the protein in a cell or otherbiological system. Both the coding strand, the nucleotide sequence ofwhich is identical to the mRNA sequence (except that a “T” is replacedby a “U”) and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

The term “exogenous” refers to a molecule that does not naturally occurin a wild-type cell or organism but is typically introduced into thecell by molecular biological techniques. Examples of exogenouspolynucleotides include vectors, plasmids, and/or man-made nucleic acidconstructs encoding the desired protein. With regard to polynucleotidesand proteins, the term “endogenous” or “native” refers tonaturally-occurring polynucleotide or amino acid sequences that may befound in a given wild-type cell or organism. Also, a particularpolynucleotide sequence that is isolated from a first organism andtransferred to a second organism by molecular biological techniques istypically considered an “exogenous” polynucleotide or amino acidsequence with respect to the second organism. In specific embodiments,polynucleotide sequences can be “introduced” by molecular biologicaltechniques into a microorganism that already contains such apolynucleotide sequence, for instance, to create one or more additionalcopies of an otherwise naturally-occurring polynucleotide sequence, andthereby facilitate overexpression of the encoded polypeptide.

The term “expression or overexpression” refers to the transcriptionand/or translation of a particular nucleotide sequence into a precursoror mature protein, for example, driven by its promoter. “Overexpression”refers to the production of a gene product in transgenic organisms orcells that exceeds levels of production in normal or non-transformedorganisms or cells. As defined herein, the term “expression” refers toexpression or overexpression.

The term “expression vector” refers to a vector including a recombinantpolynucleotide including expression control (regulatory) sequencesoperably linked to a nucleotide sequence to be expressed. An expressionvector includes sufficient cis-acting elements for expression; otherelements for expression can be supplied by the host cell or in an invitro expression system. Expression vectors include all those known inthe art, such as cosmids, plasmids (e.g., naked or contained inliposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses,and adeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “homologous” refers to sequence similarity or sequence identitybetween two polypeptides or between two polynucleotides when a positionin both of the two compared sequences is occupied by the same base oramino acid monomer subunit, e.g., if a position in each of two DNAmolecules is occupied by adenine, then the molecules are homologous atthat position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous, then the two sequences are 60% homologous. By way ofexample, the DNA sequences ATTGCC and TATGGC share 50% homology. Acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” refers to a class of proteins, whichfunction as antibodies. The five members included in this class ofproteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibodythat is present in body secretions, such as saliva, tears, breast milk,gastrointestinal secretions and mucus secretions of the respiratory andgenitourinary tracts. IgG is the most common circulating antibody. IgMis the main immunoglobulin produced in the primary immune response inmost subjects. It is the most efficient immunoglobulin in agglutination,complement fixation, and other antibody responses, and is important indefense against bacteria and viruses. IgD is the immunoglobulin that hasno known antibody function but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingthe release of mediators from mast cells and basophils upon exposure tothe allergen.

The term “isolated” refers to a material that is substantially oressentially free from components that normally accompany it in itsnative state. The material can be a cell or a macromolecule such as aprotein or nucleic acid. For example, an “isolated polynucleotide,” asused herein, refers to a polynucleotide, which has been purified fromthe sequences which flank it in a naturally-occurring state, e.g., a DNAfragment which has been removed from the sequences that are normallyadjacent to the fragment. Alternatively, an “isolated peptide” or an“isolated polypeptide” and the like, as used herein, refer to in vitroisolation and/or purification of a peptide or polypeptide molecule fromits natural cellular environment, and from association with othercomponents of the cell.

The term “substantially purified” refers to a material that issubstantially free from components that are normally associated with itin its native state. For example, a substantially purified cell refersto a cell that has been separated from other cell types with which it isnormally associated in its naturally occurring or native state. In someinstances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to a cell that has been separated from the cells with which theyare naturally associated in their natural state. In embodiments, thecells are cultured in vitro. In embodiments, the cells are not culturedin vitro.

In the context of the present disclosure, the following abbreviationsfor the commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. Moreover, the use oflentiviruses enables the integration of the genetic information into thehost chromosome, resulting in stably transduced genetic information.HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived fromlentiviruses offer the means to achieve significant levels of genetransfer in vivo.

The term “modulating,” refers to mediating a detectable increase ordecrease in the level of a response in a subject compared with the levelof a response in the subject in the absence of a treatment or compound,and/or compared with the level of a response in an otherwise identicalbut untreated subject. The term encompasses perturbing and/or affectinga native signal or response, thereby mediating a beneficial therapeuticresponse in a subject, preferably, a human.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another polynucleotide. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation.

The term “under transcriptional control” refers to a promoter beingoperably linked to and in the correct location and orientation inrelation to a polynucleotide to control (regulate) the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area such as a solid tumor withina specific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may include non-solid tumors (such as hematological tumors, forexample, leukemia, lymphoma, and multiple myeloma) or may include solidtumors. Types of cancers to be treated with the CARs of the disclosureinclude, but are not limited to, carcinoma, blastoma, and sarcoma, andcertain leukemia or lymphoid malignancies, benign and malignant tumors,and malignancies, e.g., sarcomas, carcinomas, and melanomas. Adulttumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma, and brainmetastases).

A solid tumor antigen is an antigen expressed on a solid tumor. Inembodiments, the solid tumor antigen is also expressed at low levels onhealthy tissue. Examples of solid tumor antigens and their relateddisease tumors are provided in Table 1.

TABLE 1 Solid Tumor antigen Disease tumor PRLR Breast Cancer CLCA1colorectal cancer MUC12 colorectal cancer GUCY2C colorectal cancer GPR35colorectal cancer CR1L Gastric Cancer MUC 17 Gastric Cancer TMPRSS11Besophageal cancer MUC21 esophageal cancer TMPRSS11E esophageal cancerCD207 bladder Cancer SLC30A8 pancreatic Cancer CFC1 pancreatic CancerSLC12A3 Cervical Cancer SSTR1 Cervical tumor GPR27 Ovary tumor FZD10Ovary tumor TSHR Thyroid Tumor SIGLEC15 Urothelial cancer SLC6A3 Renalcancer KISS1R Renal cancer QRFPR Renal cancer: GPR119 Pancreatic cancerCLDN6 Endometrial cancer/Urothelial cancer UPK2 Urothelial cancer(including bladder cancer) ADAM12 Breast cancer, pancreatic cancer andthe like SLC45A3 Prostate cancer ACPP Prostate cancer MUC21 Esophagealcancer MUC16 Ovarian cancer MS4A12 Colorectal cancer ALPP Endometrialcancer CEA Colorectal carcinoma EphA2 Glioma FAP Mesothelioma GPC3 Lungsquamous cell carcinoma IL13-Rα2 Glioma Mesothelin Metastatic cancerPSMA Prostate cancer ROR1 Breast lung carcinoma VEGFR-II Metastaticcancer GD2 Neuroblastoma FR-α Ovarian carcinoma ErbB2 Carcinomas EpCAMCarcinomas EGFRvIII Glioma-Glioblastoma EGFR Glioma-NSCL cancer tMUC 1Cholangiocarcinoma, Pancreatic cancer, Breast Cancer B7-H3 Ewing sarcoma(bone tumor), rhabdomyosarcoma, nephroblastoma, neuroblastoma andmedulloblastoma (brain tumor)

The term “parenteral administration” of a composition includes, e.g.,subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.),intrasternal injection, or infusion techniques.

The terms “patient,” “subject,” and “individual,” and the like are usedinterchangeably herein and refer to any human, or animal, amenable tothe methods described herein. In certain non-limiting embodiments, thepatient, subject, or individual is a human or animal. In embodiments,the term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, and animals such as dogs, cats, mice, rats, andtransgenic species thereof.

A subject in need of treatment or in need thereof includes a subjecthaving a disease, condition, or disorder that needs to be treated. Asubject in need thereof also includes a subject that needs treatment forthe prevention of a disease, condition, or disorder. In embodiments, thedisease, condition, or disorder is cancer.

The term “polynucleotide” or “nucleic acid” refers to mRNA, RNA, cRNA,rRNA, cDNA or DNA. The term typically refers to a polymeric form ofnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes all forms of nucleic acids including single anddouble-stranded forms of nucleic acids.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridize witha reference sequence under stringent conditions that are definedhereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and “variant” include polynucleotides in whichone or more nucleotides have been added or deleted or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletions,and substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide or has increased activity in relation tothe reference polynucleotide (i.e., optimized). Polynucleotide variantsinclude, for example, polynucleotides having at least 50% (and at least51% to at least 99% and all integer percentages in between, e.g., 90%,95%, or 98%) sequence identity with a reference polynucleotide sequencedescribed herein. The terms “polynucleotide variant” and “variant” alsoinclude naturally-occurring allelic variants and orthologs.

The terms “polypeptide,” “polypeptide fragment,” “peptide,” and“protein” are used interchangeably herein to refer to a polymer of aminoacid residues and to variants and synthetic analogues of the same. Thus,these terms apply to amino acid polymers in which one or more amino acidresidues are synthetic non-naturally occurring amino acids, such as achemical analogue of a corresponding naturally occurring amino acid, aswell as to naturally-occurring amino acid polymers. In certain aspects,polypeptides may include enzymatic polypeptides, or “enzymes,” whichtypically catalyze (i.e., increase the rate of) various chemicalreactions.

The term “polypeptide variant” refers to polypeptides that aredistinguished from a reference polypeptide sequence by the addition,deletion, or substitution of at least one amino acid residue. In certainembodiments, a polypeptide variant is distinguished from a referencepolypeptide by one or more substitutions, which may be conservative ornon-conservative. In certain embodiments, the polypeptide variantcomprises conservative substitutions and, in this regard, it is wellunderstood in the art that some amino acids may be changed to otherswith broadly similar properties without changing the nature of theactivity of the polypeptide. Polypeptide variants also encompasspolypeptides in which one or more amino acids have been added or deletedor replaced with different amino acid residues.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence. Theterm “expression control (regulatory) sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control (regulatory) sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilize promoters, polyadenylation signals, and enhancers.

The term “bind,” “binds,” or “interacts with” refers to a moleculerecognizing and adhering to a second molecule in a sample or organismbut does not substantially recognize or adhere to other structurallyunrelated molecules in the sample. The term “specifically binds,” asused herein with respect to an antibody, refers to an antibody whichrecognizes a specific antigen, but does not substantially recognize orbind other molecules in a sample. For example, an antibody thatspecifically binds an antigen from one species may also bind thatantigen from one or more species. But, such cross-species reactivitydoes not itself alter the classification of an antibody as specific. Inanother example, an antibody that specifically binds an antigen may alsobind different allelic forms of the antigen. However, such crossreactivity does not itself alter the classification of an antibody asspecific. In some instances, the terms “specific binding” or“specifically binding,” can be used in reference to the interaction ofan antibody, a protein, or a peptide with a second chemical species, tomean that the interaction is dependent upon the presence of a particularstructure (e.g., an antigenic determinant or epitope) on the chemicalspecies; for example, an antibody recognizes and binds a specificprotein structure rather than to any protein. If an antibody is specificfor epitope “A,” the presence of a molecule containing epitope A (orfree, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.5 or less. A “decreased” or “reduced” or“lesser” amount is typically a “statistically significant” or aphysiologically significant amount, and may include a decrease that isabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100,500, 1000 times) (including all integers and decimal points in betweenand above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or leveldescribed herein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognateligand thereby mediating a signal transduction event, such as signaltransduction via the TCR/CD3 complex. Stimulation can mediate alteredexpression of certain molecules, such as downregulation of TGF-β, and/orreorganization of cytoskeletal structures.

The term “stimulatory molecule” refers to a molecule on a T cell thatspecifically binds a cognate stimulatory ligand present on anantigen-presenting cell. For example, a functional signaling domainderived from a stimulatory molecule is the zeta chain associated withthe T cell receptor complex. The stimulatory molecule includes a domainresponsible for signal transduction.

The term “stimulatory ligand” refers to a ligand that when present on anantigen-presenting cell (e.g., an APC, a dendritic cell, a B-cell, andthe like.) can specifically bind with a cognate binding partner(referred to herein as a “stimulatory molecule”) on a cell, for examplea T cell, thereby mediating a primary response by the T cell, includingactivation, initiation of an immune response, proliferation, and similarprocesses. Stimulatory ligands are well-known in the art and encompass,inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2antibody.

The term “therapeutic” refers to treatment and/or prophylaxis. Atherapeutic effect is obtained by suppression, remission, or eradicationof a disease state or alleviating the symptoms of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or another clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent the development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, and other factors, of the subject to be treated.

The term “treat a disease” refers to the reduction of the frequency orseverity of at least one sign or symptom of a disease or disorderexperienced by a subject.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which an exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed, or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “vector” refers to a polynucleotide that comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding linear polynucleotides, polynucleotides associated with ionicor amphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. The term alsoincludes non-plasmid and non-viral compounds which facilitate thetransfer of nucleic acid into cells, such as, for example, polylysinecompounds, liposomes, and the like. Examples of viral vectors includeadenoviral vectors, adeno-associated virus vectors, retroviral vectors,and others. For example, lentiviruses are complex retroviruses, which,in addition to the common retroviral genes gag, pol, and env, containother genes with regulatory or structural function. Lentiviral vectorsare well known in the art. Some examples of lentivirus include the HumanImmunodeficiency Viruses: HIV-1, HIV-2, and the Simian ImmunodeficiencyVirus: SIV. Lentiviral vectors have been generated by multiplyattenuating the HIV virulence genes, for example, the genes env, vif,vpr, vpu, and nef are deleted making the vector biologically safe.

Ranges: throughout this disclosure, various aspects of the disclosurecan be presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

A “chimeric antigen receptor” (CAR) molecule is a recombinantpolypeptide including at least an extracellular domain, a transmembranedomain, and a cytoplasmic domain or intracellular domain. Inembodiments, the domains of the CAR are on the same polypeptide chain,for example, a chimeric fusion protein. In embodiments, the domains areon different polypeptide chains, for example, the domains are notcontiguous.

The extracellular domain of a CAR molecule includes an antigen bindingdomain. In embodiments, the antigen binding domain binds an antigen, forexample, a cell surface molecule or marker, on the surface of a B cell.In embodiments, the cell surface molecule of a B cell includes CD19,CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b,CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. In embodiments, the cellsurface molecule of the B cell is CD19, CD20, CD22, or BCMA. Inembodiments, the cell surface molecule of the B cell is CD19.

In embodiments, the antigen binding domain binds an antigen on thesurface of a tumor, for example, a tumor antigen or tumor marker. Tumorantigens are proteins that are produced by tumor cells that elicit animmune response, particularly T cell-mediated immune 0

In embodiments, the extracellular antigen binding domain of a CARincludes at least one scFv or at least a single domain antibody. As anexample, there can be two scFvs on a CAR. The scFv includes a lightchain variable (VL) region and a heavy chain variable (VH) region of atarget antigen-specific monoclonal antibody joined by a flexible linker.Single chain variable region fragments can be made by linking lightand/or heavy chain variable regions by using a short linking peptide(Bird et al., Science 242:423-426, 1988). An example of a linkingpeptide is the GS linker having the amino acid sequence (GGGGS)3 (SEQ IDNO: 2), which bridges approximately 3.5 nm between the carboxy terminusof one variable region and the amino terminus of the other variableregion. Linkers of other sequences have been designed and used (Bird etal., 1988, supra). In general, linkers can be short, flexiblepolypeptides comprising about 20 or fewer amino acid residues. Thesingle-chain variants can be produced either recombinantly orsynthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

In embodiments, the CAR molecules described herein comprises one or moreCDRs for binding an antigen of interest, for example, one or more CDRsfor binding CD19 or tMUC1.

The cytoplasmic domain of the CAR molecules described herein includesone or more co-stimulatory domains and one or more signaling domains.The co-stimulatory and signaling domains function to transmit the signaland activate molecules, such as T cells, in response to antigen binding.The one or more co-stimulatory domains are derived from stimulatorymolecules and/or co-stimulatory molecules, and the signaling domain isderived from a primary signaling domain, such as the CD3 zeta domain. Inembodiments, the signaling domain further includes one or morefunctional signaling domains derived from a co-stimulatory molecule. Inembodiments, the co-stimulatory molecules are cell surface molecules(other than antigens receptors or their ligands) that are required foractivating a cellular response to an antigen.

In embodiments, the co-stimulatory domain includes the intracellulardomain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds CD83, or any combination thereof. Inembodiments, the signaling domain includes a CD3 zeta domain derivedfrom a T cell receptor.

In embodiments, the cytoplasmic domain of the CAR only includes one ormore stimulatory domains and no signaling domain.

The CAR molecules also include a transmembrane domain. The incorporationof a transmembrane domain in the CAR molecules stabilizes the molecule.In embodiments, the transmembrane domain of the CAR molecules is thetransmembrane domain of a CD28 or 4-1BB molecule.

Between the extracellular domain and the transmembrane domain of theCAR, there may be incorporated a spacer domain. As used herein, the term“spacer domain” generally means any oligo- or polypeptide that functionsto link the transmembrane domain to, either the extracellular domain or,the cytoplasmic domain on the polypeptide chain. A spacer domain mayinclude up to 300 amino acids, preferably 10 to 100 amino acids, andmost preferably 25 to 50 amino acids.

CAR Molecule(s) and Therapeutic Agent(s)

The present disclosure describes a cell modified to express one or moremolecules at a level that is higher or lower than the level of the oneor more molecules expressed by a cell that has not been modified toexpress the one or more molecules, wherein the one or more molecules areassociated with cell migration. Embodiments also describe a modifiedcell engineered to express an antigen binding molecule, whereinexpression and/or function of one or more molecules in the modified cellhas been enhanced or reduced, wherein the one or more molecules areassociated with cell migration. In embodiments, the modified cellcomprises a disruption in an endogenous gene or an addition of exogenousgene that is associated with a biosynthesis or transportation pathway ofthe one or more molecules. As used herein, the term “associated withcell migration” refers to being involved with cell migration. As anexample, “a molecule associated with cell migration” means that themolecule can activate, induce, disrupt, or inhibit cell migration.

The terms “trafficking ability” and “migration ability” of cells and thelike are used interchangeably herein and refer to the ability orcapability of cells (e.g., T cells) migration in response to, forexample, chemokines and stimulation (e.g., tumor environment). Examplesof the chemokines may include CCL1, CCL5, CCL2, CCL22, CCL17, CXCL9, andCXCL10, CXCL11. For example, modified T cells overexpressing CDC42 orhaving reduced expression of LRCH1 show enhanced migration ability inthe migration assay. As shown in the Example below, the number of themodified cells migrate or traffic to the medium containing CCL5 isgreater than that of T cells neither overexpressing CDC42 nor havingreduced expression of LRCH1.

Embodiments described herein relate to a method or use ofpolynucleotide, the method comprising providing a viral particle (e.g.,AAV, lentivirus or their variants) comprising a vector genome, thevector genome comprising the polynucleotide encoding the one moremolecules and a polynucleotide encoding a binding molecule, thepolynucleotide operably linked to an expression control elementconferring transcription of the polynucleotides; and administering anamount of the viral particle to a subject such that the polynucleotideis expressed in the subject, where the one or more molecules areassociated with cell migration. In embodiments, the AAV preparation mayinclude AAV vector particles, empty capsids and host cell impurities,thereby providing an AAV product substantially free of AAV emptycapsids.

Embodiments described herein relate to a pharmaceutical compositioncomprising the population of the modified cells described herein.Embodiments also describe a method of causing or eliciting T cellresponse in a subject in need thereof and/or treating a tumor of thesubject, the method comprising administering an effective amount of thepharmaceutical composition to the subject. Embodiments described hereinalso relate to an isolated nucleic acid sequence encoding one or moremolecules that are associated with cell migration.

In embodiments, the one or more molecules comprise at least one ofDOCK8, CDC42, and LRCH1, a functional variant of the one or moremolecules, or a functional fragment of the one or more molecules. Inembodiments, the one or more molecules are or comprise CDC42, which isoverexpressed in the modified T cells. In embodiments, the one or moremolecules are or comprise LRCH1 of which expression is reduced in themodified T cells. In embodiments, the modified cell comprises arecombinant polynucleotide encoding SEQ ID NOS: 43 and/or 39. Inembodiments, the modified cell comprises a recombinant polynucleotidecomprising SEQ ID NO: 38. In embodiments, the modified cell comprises arecombinant polynucleotide encoding SEQ ID NOS: 43 and encoding anantigen binding molecule (e.g., CAR). In embodiments, the modified cellcomprises a recombinant polynucleotide comprising SEQ ID NO: 38 andencoding an antigen binding molecule (e.g., CAR). In embodiments, themodified cell comprises at least one of SEQ ID Nos: 44-47.

Cell division control protein 42 homolog, also known as Cdc42, is aprotein involved in regulation of the cell cycle. It was originallyidentified in S. cerevisiae (yeast) as a mediator of cell division andis now known to influence a variety of signaling events and cellularprocesses in a variety of organisms from yeast to mammals. DOCK8(Dedicator of cytokinesis 8), also known as Zir3, is a large (˜190 kDa)protein involved in intracellular signaling networks and is a member ofthe DOCK-C subfamily of the DOCK family of guanine nucleotide exchangefactors (GEFs) which function as activators of small G proteins.Leucine-rich repeat and calponin homology domain-containing protein 1(LRCH1) includes a leucine-rich repeat and a calponin homology domain,and function as a negative regulator of GTPase CDC42 by sequesteringCDC42-guanine exchange factor DOCK8, probably by preventing CDC42activation and negatively regulates CD4+ T-cell migration.

In embodiments, the polynucleotide may integrate into the genome of themodified cell, and descendants of the modified cell will also expressthe polynucleotide, resulting in a stably transfected modified cell. Inembodiments, the modified cell may express the polynucleotide encodingthe CAR but the polynucleotide does not integrate into the genome of themodified cell such that the modified cell expresses the transientlytransfected polynucleotide for a finite period of time, for example afew days, after which the polynucleotide is lost through cell divisionor other cellular processes. As an example, the polynucleotide ispresent in the modified cell in a recombinant DNA construct, in an mRNA,or in a viral vector, and/or the polynucleotide is an mRNA, which is notintegrated into the genome of the modified cell.

Embodiments relate to a method or use of the polynucleotides describedherein. The method or use includes: providing a viral particle (e.g.,AAV, lentivirus or their variants) comprising a vector genome, thevector genome comprising the polynucleotide, wherein the polynucleotideis operably linked to an expression control element conferringtranscription of the polynucleotide; and administering an amount of theviral particle to the subject such that the polynucleotide is expressedin the subject. In embodiments, the AAV preparation includes AAV vectorparticles, empty capsids, and host cell impurities, thereby providing anAAV product substantially free of AAV empty capsids. More information ofthe administration and preparation of the viral particle may be found atthe U.S. Pat. No. 9,840,719 and Milani et al., Sci. Transl. Med. 11,eaav7325 (2019) 22 May 2019, which are incorporated herein by reference.

In embodiments, a bioreactor can be inoculated at a cell density ofapproximately 0.5×10⁶ cells/mL with viability greater than 95%. When thecell density reaches approximately 1.0×10⁶cells/mL, the cells may betransfected with the polyethyleneimine (PEI)/DNA complexes (polyplexes)with a PEI to DNA ratio of 2:1. At the time of harvest, AAV from thecell culture in the bioreactor may be released using the Triton X-100method. All solutions may be added directly to the bioreactor, and thelysate centrifuged at 4000×g for 20 min. The supernatant can be storedat −80 C for further processing. AAV may be further purified. Forexample, AAV samples (12.3 mL) may be purified by overlaying them on topof a series of step gradients using 15, 25, 40, and 54% iodixanolconcentrations containing 1, 5, 7, and 5 mL, respectively. The 15%iodixanol concentration also contains 1 M NaCl to avoid aggregation ofAAV with other cellular proteins and negatively charged nuclearcomponents. After the completion of centrifugation, 5 mL may bewithdrawn from 2 mm below the 40/54 interface marked before starting theultracentrifugation at 385,000×g for 1 h 45 min in Sorvals T-865 rotorin Sorval Ultracentrifuge. The viral vectors can then be quantified. Forexample, vectors AAV infectivity can be determined by the gene transferassay (GTA) using GFP as a reporter gene in all cases. AAV infectivityassay, in which samples are diluted before addition to the cells, havethe GFP positive cells in the range of 2-20% to ensure that only asingle virus has entered the cell for GFP expression. The GFP-positivecells may be quantified by FACS using HEK293 cells in suspension. TheAAV may be then administrated to a subject. For example, AAV may bediluted in 0.9% sterile NaCl saline solution (supplemented with 0.25%human serum albumin [HSA]) for infusion in patients, and the finalvolume of infusion can be calculated based on the patient's weight as 3mL/kg.

In embodiments, the modified cell comprises the antigen bindingmolecule, and the antigen binding molecule is a CAR, which comprises anantigen-binding domain, a transmembrane domain, and an intracellularsignaling domain. In embodiments, the antigen-binding domain binds atumor antigen selected from the group consisting of: TSHR, CD19, CD123,CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag,PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT,IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24,PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1,EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2,gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGSS, HMWMAA,o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D,CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1,UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1,LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8,MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints,ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor,Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK,AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2,intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1,FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, andIGLL1. In embodiments, the intracellular signaling domain comprises aco-stimulatory signaling domain, or a primary signaling domain and aco-stimulatory signaling domain, wherein the co-stimulatory signalingdomain comprises a functional signaling domain of a protein selectedfrom the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160,CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4,VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d,ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1,CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226),SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D

In embodiments, the modified cell comprises the antigen bindingmolecule, the antigen binding molecule is a modified TCR. Inembodiments, the TCR is derived from spontaneously occurringtumor-specific T cells in patients. In embodiments, the TCR binds to atumor antigen. In embodiments, the tumor antigen comprises CEA, gp100,MART-1, p53, MAGE-A3, or NY-ESO-1. In embodiments, the TCR comprisesTCRγ and TCRδ Chains or TCRα and TCRβ chains, or a combination thereof.

In embodiments, the cell is an immune effector cell. (e.g., a populationof immune effector cells). In embodiments, the immune effector cell is aT cell or an NK cell. In embodiments, the immune effector cell is a Tcell. In embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or acombination thereof. In embodiments, the cell is a human cell.

In embodiments, the enhanced expression and/or function of the one ormore molecules are implemented by introducing a nucleic acid sequenceencoding the one or more molecules and/or the binding molecule, which ispresent in the modified cell in a recombinant DNA construct, in an mRNA,or in a viral vector. In embodiments, the nucleic acid sequence is anmRNA, which is not integrated into the genome of the modified cell. Inembodiments, the nucleic acid sequence is associated with anoxygen-sensitive polypeptide domain. In embodiments, theoxygen-sensitive polypeptide domain comprises HIF VHL binding domain. Inembodiments, the nucleic acid sequence is regulated by a promotercomprising a binding site for a transcription modulator that modulatesthe expression of the CDC42 in the cell. In embodiments, thetranscription modulator is or includes Hif1a, NFAT, FOXP3, and/or NFkB.

In embodiments, expression of one or more therapeutic agents may beregulated by an inducible expression system. Examples of therapeuticagents includes cytokines. The inducible expression system allows for atemporal and spatial controlled activation and/or expression of genes.For example, Tetracycline-Controlled Transcriptional Activation is amethod of inducible gene expression where transcription is reversiblyturned on or off in the presence of the antibiotic tetracycline or oneof its derivatives (e.g., doxycycline). For example, an induciblesuicide gene expression system allows for a temporal and spatialcontrolled activation and/or expression of a suicide gene, which causesa cell to kill itself through apoptosis.

In embodiments, the modified cells comprise a nucleic acid sequenceencoding a reverse tetracycline transactivator (rtTA). In embodiments,expression of one or more therapeutic agents is regulated by the rtTA,such that the one or more therapeutic agents are expressed in thepresence of tetracycline. In embodiments, a concentration oftetracycline in the cell culture medium is not less than about 2 μg/ml.In embodiments, the tetracycline is selected from the group consistingof tetracycline, demeclocycline, meclocycline, doxycycline, lymecycline,methacycline, minocycline, oxytetracycline, rolitetracycline, andchlortetracycline. In embodiments, the tetracycline is doxycycline.

In embodiments, the inducible suicide system is an HSV-TK system or aninducible caspase-9 system. In embodiments, the modified cells comprisea nucleic acid sequence encoding a suicide gene, such that when themodified cells are in the presence of a nucleoside analogue in a mannerpermitting expression of the suicide gene, to render the nucleosideanalogue cytotoxic to the modified cells. In embodiments, the suicidegene is selected from the group consisting of thymidine kinase of herpessimplex virus, thymidine kinase of varicella zoster virus, and bacterialcytosine deaminase. In embodiments, the suicide gene is thymidine kinaseof herpes simplex virus. In embodiments, the nucleoside analogue isselected from the group consisting of ganciclovir, acyclovir,buciclovir, famciclovir, penciclovir, valciclovir, trifluorothymidine,1-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A,araT 1-beta-D-arabinofuranoxyl thymine, 5-ethyl-2′-deoxyuridine,5-iodo-5′-amino-2,5′-dideoxyuridine, idoxuridine, AZT, AIU,dideoxycytidine, and AraC. In embodiments, the nucleoside analogue isganciclovir.

Embodiments relate to a modified T cell comprising an antigen bindingmolecule, wherein expression and/or function of CDC42 in the modifiedcell has been enhanced. In embodiments, the modified cell has anenhanced T cell response. For example, the T cell response may bemeasured based on a level of cytokine releases, a level of proteasereleases, and/or migration capability. In embodiments, the modified cellhas an increased level of cytokine release in response to an antigenthat the antigen binding molecule binds as compared to a corresponding Tcell that does not overexpress CDC42. In embodiments, the cytokinerelease comprises a cytokine release of IFNγ. In embodiments, theprotease release comprises a protease release of Granzyme B (GRAM B).Granzyme B is a serine protease most commonly found in the granules ofnatural killer cells (NK cells) and cytotoxic T cells. It is secreted bythese cells along with the pore forming protein perforin to mediateapoptosis in target cells. Granzyme B has also more recently found to beproduced by a wide range of non-cytotoxic cells ranging from basophilsand mast cells to smooth muscle cells. The secondary functions ofgranzyme B are also numerous. Granzyme B has shown to be involved ininducing inflammation by stimulating cytokine release and is alsoinvolved in extracellular matrix remodeling. In embodiments, themodified cell has an enhanced migration capability in response to achemokine as compared to a corresponding T cell that does notoverexpress CDC42. In embodiments, the chemokine is CCL5. Inembodiments, the modified cell comprises a polynucleotide encoding SEQID NO: 43 and the antigen binding molecule. In embodiments, the modifiedcell comprises a polynucleotide encoding SEQ ID NO: 43, thepolynucleotide is present in the modified cell in a recombinant DNAconstruct, in an mRNA, or in a viral vector. In embodiments, thepolynucleotide is regulated by a promoter comprising a binding site fora transcription modulator that modulates the expression of the CDC42 inthe cell. In embodiments, the transcription modulator is or includesHif1a, NFAT, FOXP3, and/or NFkB. Embodiments relate to an isolatednucleic acid comprising the polynucleotide.

Modified T-cells can be derived from a stem cell. The stem cells can beadult stem cells, embryonic stem cells, more particularly non-human stemcells, cord blood stem cells, progenitor cells, bone marrow stem cells,induced pluripotent stem cells, totipotent stem cells or hematopoieticstem cells. A modified cell can also be a dendritic cell, an NK-cell, aB-cell, or a T-cell selected from the group consisting of inflammatoryT-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes orhelper T-lymphocytes. In another embodiment, Modified cells can bederived from the group consisting of CD4+T-lymphocytes andCD8+T-lymphocytes. Prior to expansion and genetic modification of thecells of the invention, a source of cells can be obtained from a subjectthrough a variety of non-limiting methods. T cells can be obtained froma number of non-limiting sources, including peripheral blood mononuclearcells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissuefrom a site of infection, ascites, pleural effusion, spleen tissue, andtumors. In embodiments, any number of T cell lines available and knownto those skilled in the art can be used. In embodiments, modified cellscan be derived from a healthy donor, from a patient diagnosed withcancer or from a patient diagnosed with an infection. In embodiments,modified cell is part of a mixed population of cells which presentdifferent phenotypic characteristics.

The term “stem cell” refers to any of certain type of cells which havethe capacity for self-renewal and the ability to differentiate intoother kinds of cells. For example, a stem cell gives rise either to twodaughter stem cells (as occurs in vitro with embryonic stem cells inculture) or to one stem cell and a cell that undergoes differentiation(as occurs, e.g. in hematopoietic stem cells, which give rise to bloodcells). Different categories of stem cells may be distinguished on thebasis of their origin and/or the extent of their capacity fordifferentiation into other types of cells. For example, stem cells mayinclude embryonic stem (ES) cells (i.e., pluripotent stem cells),somatic stem cells, Induced pluripotent stem cells, and any other typesstem cells.

The pluripotent embryonic stem cells may be found in the inner cell massof a blastocyst and have a high innate capacity for differentiation. Forexample, pluripotent embryonic stem cells may have the potential to formany type of cell in the body. When grown in vitro for long periods oftime, ES cells maintain pluripotency: progeny cells retain the potentialfor multilineage differentiation.

Somatic stem cells include the fetal stem cells (from the fetus) andadult stem cells (found in various tissues, such as bone marrow). Thesecells have been regarded as having a capacity for differentiation lowerthan that of the pluripotent ES cells—with the capacity of fetal stemcells being greater than that of adult stem cells; they apparentlydifferentiate into only a few types of cells and have been described asmultipotent. The ‘tissue-specific’ stem cells normally give rise to onlyone type of cell. For example, embryonic stem cells may bedifferentiated into blood stem cells (e.g., Hematopoietic stem cells(HSCs)), which may be further differentiated into various blood cells(e.g., red blood cells, platelets, white blood cells).

Induced pluripotent stem cells (i.e., iPS cells or iPSCs) may include atype of pluripotent stem cell artificially derived from anon-pluripotent cell (e.g., an adult somatic cell) by inducing anexpression of specific genes. Induced pluripotent stem cells are similarto natural pluripotent stem cells, such as embryonic stem (ES) cells, inmany aspects, such as the expression of certain stem cell genes andproteins, chromatin methylation patterns, doubling time, embryoid bodyformation, teratoma formation, viable chimera formation, and potency anddifferentiability. Induced pluripotent cells can be made from adultstomach, liver, skin cells, and blood cells.

In embodiments, the two different antigen binding domains can be on aCAR, and a T cell receptor (TCR) and are encoded by separate nucleicacids. The binding domain of a TCR can target a specific tumor antigenor tumor marker on the cell of a tumor. In embodiments, the TCR bindingdomain is a TCR alpha binding domain or TCR beta binding domain thattargets a specific tumor antigen. In embodiments, the TCR comprises theTCRγ and TCRδ chains or the TCRα and TCRβ chains. The present disclosurealso describes vectors including the isolated nucleic acids describedherein. In embodiments, a single vector contains the isolated nucleicacid encoding the first CAR and second CAR or TCR. In embodiments, afirst vector contains the first nucleic acid encoding a first CAR, and asecond vector contains the nucleic acid encoding the second CAR or TCR.In embodiments, the vector comprises a bispecific CAR including at leasttwo antigen binding domains.

Lymphocyte or T cell response in a subject refers to cell-mediatedimmunity associated with a helper, killer, regulatory, and other typesof T cells. For example, T cell response may include activities such asassisting other WBCs in immunologic processes and identifying anddestroying virus-infected cells and tumor cells. T cell response in thesubject can be measured via various indicators such as a number ofvirus-infected cells and/or tumor cells that T cells kill, the amount ofcytokine that T cells release in co-culturing with virus-infected cellsand/or tumor cells, a level of proliferation of T cells in the subject,a phenotype change of T cells, for example, changes to memory T cells,and a level longevity or lifetime of T cells in the subject.

In embodiments, the method of enhancing T cell response comprisestreating a subject in need thereof, for example, a subject diagnosedwith a tumor. The term tumor refers to a mass, which can be a collectionof fluid, such as blood, or a solid mass. A tumor can be malignant(cancerous) or benign. Examples of blood cancers include chroniclymphocytic leukemia, acute myeloid leukemia, acute lymphoblasticleukemia, and multiple myeloma.

Solid tumors usually do not contain cysts or liquid areas. The majortypes of malignant solid tumors include sarcomas and carcinomas.Sarcomas are tumors that develop in soft tissue cells called mesenchymalcells, which can be found in blood vessels, bone, fat tissues, ligamentlymph vessels, nerves, cartilage, muscle, ligaments, or tendon, whilecarcinomas are tumors that form in epithelial cells, which are found inthe skin and mucous membranes. The most common types of sarcomas includeundifferentiated pleomorphic sarcoma, which involves soft tissue andbone cells; leiomyosarcoma, which involves smooth muscle cells that lineblood vessels, gastrointestinal tract, and uterus; osteosarcoma whichinvolves bone cells, and liposarcoma which involves fat cells. Someexamples of sarcomas include Ewing sarcoma, Rhabdomyosarcoma,chondosarcoma, mesothelioma, fibrosarcoma, fibrosarcoma, and glioma.

The five most common carcinomas include adrenocarcinoma which involvesorgans that produce fluids or mucous, such as the breasts and prostate;basal cell carcinoma which involves cells of the outer-most layer of theskin, for example, skin cancer; squamous cell carcinoma which involvesthe basal cells of the skin; and transitional cell carcinoma whichaffects transitional cells in the urinary tract which includes thebladder, kidneys, and ureter. Examples of carcinomas include cancers ofthe thyroid, breast, prostate, lung, intestine, skin, pancreas, liver,kidneys, and bladder, and cholangiocarcinoma.

The methods described herein can be used to treat a subject diagnosedwith cancer. Cancer can be a blood cancer or can be a solid tumor, suchas a sarcoma or carcinoma. The method of treating includes administeringan effective amount of T cells comprising a first antigen binding domainand a second antigen binding domain to the subject to provide a T-cellresponse, wherein the first antigen binding domain binds a cell surfacemolecule of a WBC, and the second antigen binding domain binds anantigen different from the cell surface molecule of the WBC. Inembodiments, enhancing the T cell response in the subject includesselectively enhancing proliferation of T cell expressing the firstantigen binding domain and the second antigen binding domain in vivo.

In embodiments, the first antigen binding domain is on a CAR, and thesecond antigen binding domain is on a T Cell Receptor (TCR). Inembodiments, the TCR is a modified TCR. In embodiments, the TCR isderived from spontaneously occurring tumor-specific T cells in patients.In embodiments, the TCR binds a tumor antigen. In embodiments, the tumorantigen comprises CEA, gp100, MART-1, p53, MAGE-A3, or NY-ESO-1.

In embodiments, a T cell clone that expresses a TCR with a high affinityfor the target antigen may be isolated. Tumor-infiltrating lymphocytes(TILs) or PBMCs can be cultured in the presence of antigen-presentingcells (APCs) pulsed with a peptide representing an epitope known toelicit a dominant T cell response when presented in the context of adefined HLA allele. High-affinity clones may be then selected on thebasis of MHC—peptide tetramer staining and/or the ability to recognizeand lyse target cells pulsed with low titrated concentrations of cognatepeptide antigen. After the clone has been selected, the TCRα and TCRβchains or TCRγ and TCRδ chains are identified and isolated by molecularcloning. For example, for TCRα and TCRβ chains, the TCRα and TCRβ genesequences are then used to generate an expression construct that ideallypromotes stable, high-level expression of both TCR chains in human Tcells. The transduction vehicle, for example, a gammaretrovirus orlentivirus, can then be generated and tested for functionality (antigenspecificity and functional avidity) and used to produce a clinical lotof the vector. An aliquot of the final product can then be used totransduce the target T cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

Various methods may be implemented to obtain genes encodingtumor-reactive TCR. More information is provided in Kershaw et al., ClinTransl Immunology. 2014 May; 3(5): e16. In embodiments, specific TCR canbe derived from spontaneously occurring tumor-specific T cells inpatients. Antigens included in this category include the melanocytedifferentiation antigens MART-1 and gp100, as well as the MAGE antigensand NY-ESO-1, with expression in a broader range of cancers. TCRsspecific for viral-associated malignancies can also be isolated, as longas viral proteins are expressed by transformed cells. Malignancies inthis category include liver and cervical cancer, associated withhepatitis and papilloma viruses, and Epstein-Barr virus-associatedmalignancies. In embodiments, target antigens of the TCR may include CEA(e.g., for colorectal cancer), gp100, MART-1, p53 (e.g., for Melanoma),MAGE-A3 (e.g., Melanoma, esophageal and synovial sarcoma), NY-ESO-1(e.g., for Melanoma and sarcoma as well as Multiple myelomas).

In embodiments, preparation and transfusion of tumor infiltratinglymphocytes (TIL) may be implemented by the following. For example,tumor tissue from surgical or biopsy specimens can be obtained underaseptic conditions and transported to the cell culture chamber in theice box. Necrotic tissue and adipose tissue can be removed. The tumortissue may be cut into small pieces of about 1-3 cubic millimeters.Collagenase, hyaluronidase, and DNA enzyme can be added and digestedovernight at 4° C. Filtering with 0.2 um filter, cells can be separatedand collected by lymphocyte separation fluid, 1500 rpm for 5 min.Expanding the cells with a culture medium comprising PHA,2-mercaptoethanol, and CD3 monoclonal antibody, a small dose of IL-2(10-20 IU/ml) can be added to induce activation and proliferation.According to the growth situation, the cell density can be carefullydetected and maintained within the range of 0.5-2×10⁶/ml under thecondition of 37° C. and 5% CO2 for 7-14 days. TIL positive cells havethe ability to kill homologous cancer cell may be screened out byco-culture. The positive cells can be amplified in a serum-free mediumcontaining a high dose of IL2 (5000-6000 IU/ml) until greater than1×10¹¹ TILs can be obtained. To administer TILs, they may be firstcollected in saline solution using continuous-flow centrifugation andthen filtered through a platelet-administration set into a volume of200-300 ml containing 5% albumin and 450 000 IU of IL-2. The TILs may beinfused into patients through a central venous catheter over a period of30-60 minutes. In embodiments, TILs may be infused in two to fourseparate bags; the infusions may be separated by several hours.

In embodiments, the isolated T cell comprises a dominant negativevariant of a receptor of programmed death 1 (PD-1), cytotoxic Tlymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), Tcell immunoglobulin mucin-3 (TIM-3), lymphocyte-activation protein 3(LAG-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT),leukocyte-associated immunoglobulin-like receptor 1 (LAIRI), naturalkiller cell receptor 2B4 (2B4), or CD 160. In embodiments, the isolatedT cell comprises a reduced amount of TCR, as compared to thecorresponding wide-type T cell. Dominant negative mutations have analtered gene product that acts antagonistically to the wild-type allele.These mutations usually result in an altered molecular function (ofteninactive) and are characterized by a dominant or semi-dominantphenotype.

The present disclosure describes pharmaceutical compositions. Thepharmaceutical compositions include one or more of the following: CARmolecules, TCR molecules, modified CAR T cells, modified cellscomprising CAR or TCR, modified cells, nucleic acids, and vectorsdescribed herein. Pharmaceutical compositions are administered in amanner appropriate to the patient based on disease being treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “a tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentdisclosure to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can be stated that a pharmaceutical composition comprisingthe T cells described herein may be administered at a dosage of 10⁴ to10⁹cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. T cell compositionsmay also be administered multiple times at these dosages. The cells canbe administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly. In certain embodiments, it may be desired toadminister activated T cells to a subject and then subsequently redrawthe blood (or have apheresis performed), collect the activated andexpanded T cells, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocols, may select out certainpopulations of T cells.

The administration of the pharmaceutical compositions described hereinmay be carried out in any convenient manner, including by aerosolinhalation, injection, ingestion, transfusion, implantation, ortransplantation. The compositions described herein may be administeredto a patient subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i. v.)injection, or intraperitoneally. In embodiments, the T cell compositionsdescribed herein are administered to subjects by intradermal orsubcutaneous injection. In embodiments, the T cell compositions of thepresent disclosure are administered by i.v. injection. The compositionsof T cells may be injected directly into a tumor, lymph node, or site ofinfection. In embodiments, cells activated and expanded using themethods described herein, or other methods known in the art where Tcells are expanded to therapeutic levels, are administered to patientsin conjunction with (e.g., before, simultaneously or following) anynumber of relevant treatment modalities, including but not limited totreatment with agents for antiviral therapy, cidofovir andinterleukin-2, Cytarabine (also known as ARA-C); or natalizumabtreatment for MS patients; or efalizumab treatment for psoriasispatients or other treatments for PML patients. In further embodiments,the T cells described herein can be used in combination withchemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAM PATH, anti-CD3 antibodies orother antibody therapies, cytoxin, fludaribine, cyclosporin, FK506,rapamycin, mycophenolic acid, steroids, FR901228, cytokines, andirradiation. These drugs inhibit either the calcium dependentphosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993;Isoniemi (supra)). In embodiments, the cell compositions describedherein are administered to a subject in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In embodiments, the cell compositions describedherein are administered following B-cell ablative therapy. For example,agents that react with CD20, e.g., Rituxan may be administered topatients. In embodiments, subjects may undergo standard treatment withhigh dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentdisclosure. In embodiments, expanded cells are administered before orfollowing surgery.

In embodiments, CpG oligonucleotides (e.g., Class B CpGoligonucleotides) can be systemically and repeatedly administered to asubject to enhance anti-tumor effect of the pharmaceuticals describedherein (e.g., CAR T cells) by introducing macrophage activation. Forexample, the administration of CAR T cells and CpG oligonucleotides canbe combined to treat a subject having a solid tumor. Information onadministration of CpG oligonucleotides may be found at Nat Immunol. 2019March; 20(3): 265-275, which is incorporated by reference in itsentirety.

The dosage of the above treatments to be administered to a subject inneed thereof will vary with the precise nature of the condition beingtreated and the recipient of the treatment. The scaling of dosages forhuman administration can be performed according to art-acceptedpractices by a physician depending on various factors.

Additional information on the methods of cancer treatment usingengineered or modified T cells is provided in U.S. Pat. No. 8,906,682,incorporated by reference in its entirety.

Embodiments described herein relate to an in vitro method for preparingmodified cells. The method may include obtaining a sample of cells froma subject. For example, the sample may include T cells or T cellprogenitors. The method may further include transfecting the sample ofcells with a DNA encoding at least a CAR and culturing the population ofCAR cells ex vivo in a medium that selectively enhances proliferation ofCAR-expressing T cells.

In embodiments, the sample is a cryopreserved sample. In embodiments,the sample of cells is from umbilical cord blood or a peripheral bloodsample from the subject. In embodiments, the sample of cells is obtainedby apheresis or venipuncture. In embodiments, the sample of cells is asubpopulation of T cells.

The present disclosure is further described by reference to thefollowing exemplary embodiments and examples. These exemplaryembodiments and examples are provided for purposes of illustration onlyand are not intended to be limiting unless otherwise specified. Thus,the present disclosure should in no way be construed as being limited tothe following exemplary embodiments and examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Exemplary Embodiments

The following are exemplary embodiments:

1. An isolated nucleic acid sequence comprising a nucleic acid sequenceand an additional nucleic acid sequence, the nucleic acid sequenceencoding an antigen binding molecule, the additional nucleic acidsequence being associated with a disruption in an endogenous gene orencoding at least one of LRCH1, DOCK8, and Cdc42.2. A modified cell comprising: an antigen binding molecule; and:a disruption in an endogenous gene, oran addition of exogenous gene that is associated with a biosynthesis ortransportation pathway of at least one of LRCH1, DOCK8, and Cdc42,wherein the modified cell has an increased amount of DOCK8 or Cdc42and/or a reduced amount of LRCH1 as compared with a corresponding wildtype cell.3. The modified cell of embodiment 2, wherein the modified T cell has adisruption in an endogenous gene associated with a biosynthesis ortransportation pathway of LRCH1.4. The modified cell of embodiment 3, further comprising a TALENtargeting SEQ ID NO: 24.5. The modified cell of embodiment 3, wherein the TALEN comprises a leftarm comprising SEQ ID NO: 34 and a right arm comprising SEQ ID NO: 35.6. The modified cell of embodiment 3, further comprising a ZFN targetingSEQ ID NO: 23.7. The modified cell of embodiment 3, wherein the ZFN comprises a leftarm comprising one of the nucleic acid sequences of SEQ ID NOs: 26-29and a right arm comprising one of the nucleic acid sequences of SEQ IDNOs: 30-33.8. The modified cell of embodiment 2, wherein DOCK8 or Cdc42 isoverexpressed as compared to the corresponding wild type cell.9. The modified cell of embodiment 2, wherein a level of expression ofDOCK8 or Cdc42 is greater than the average level of expression of DOCK8or Cdc42 on the cell by at least about 10%, 20%, 30%, 40%, or 50%.10. The modified cell of embodiment 2, wherein the genome of the cellcomprises a polynucleotide sequence encoding the DOCK8 or Cdc42, thepolynucleotide sequence operably linked to a promoter polynucleotidesequence.11. The modified cell of embodiment 2, wherein the modified cell is alymphocyte, leukocyte, or PBMC.12. A pharmaceutical composition comprising a population of the modifiedcells of any of embodiments 2-11.13. A method of causing or eliciting a T cell response in a subject inneed thereof and/or treating a tumor of the subject, the methodcomprising administering an effective amount of the composition ofembodiment 12 to the subject.14. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 1-11, wherein the binding molecule is a CARthat comprises an extracellular domain, a transmembrane domain, and anintracellular domain, and the extracellular domain binds an antigen.15. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 14, wherein the intracellular domain comprises acostimulatory signaling region that comprises an intracellular domain ofa costimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and any combination thereof.16. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 15, wherein the antigen is Epidermal growth factorreceptor (EGFR), Variant III of the epidermal growth factor receptor(EGFRvIII), Human epidermal growth factor receptor 2 (HER2), Mesothelin(MSLN), Prostate-specific membrane antigen (PSMA), Carcinoembryonicantigen (CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2),Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesionmolecule (L1-CAM), Cancer antigen 125 (CA125), Cluster ofdifferentiation 133 (CD133), Fibroblast activation protein (FAP),Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α(FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-CellMaturation Antigen (BCMA), or CD4.17. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-16, wherein the modified cell comprises adominant negative PD-1 mutant such that PD-1/PDI-1 signaling pathway ofthe cell is interfered.18. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-17, wherein the DOCK8 or Cdc42 is present inthe modified cell in a recombinant DNA construct, in an mRNA, or in aviral vector.19. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-18, wherein the modified cell comprises aDOCK8 or Cdc42 mRNA encoding the DOCK8 or Cdc42, and the mRNA is notintegrated into the genome of the modified cell.20. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-19, wherein the modified cell comprises anucleic acid sequence comprising or the isolated nucleic acid sequencecomprises a promoter comprising a binding site for a transcriptionmodulator that modulates expression of a therapeutic agent in the cell.21. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 20, wherein the transcription modulator is or includesHif1a, N FAT, FOXP3, and/or NFkB.22. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 21, wherein the promoter is responsive to thetranscription modulator.23. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 22, wherein the promoter is operably linked to the nucleicacid sequence encoding the therapeutic agent such that the promoterdrives expression of the therapeutic agent in the cell.24. A cell modified to express one or more molecules at a level that ishigher or lower than the level of the one or more molecules expressed bya cell that has not been modified to express the one or more molecules,wherein the one or more molecules are associated with cell migration.25. A modified cell engineered to express an antigen binding molecule,wherein expression and/or function of one or more molecules in themodified cell has been enhanced or reduced, and wherein the one or moremolecules are associated with cell migration.26. The modified cell of any one of embodiments 24 or 25, wherein themodified cell comprises: a disruption in an endogenous gene, or anaddition of exogenous gene that is associated with a biosynthesis ortransportation pathway of the one or more molecules.27. A method or use of polynucleotide, the method comprising providing aviral particle (e.g., AAV, lentivirus or their variants) comprising avector genome, the vector genome comprising the polynucleotide encodingthe one more molecules and a polynucleotide encoding a binding molecule,the polynucleotide operably linked to an expression control elementconferring transcription of the polynucleotides; and administering anamount of the viral particle to a subject such that the polynucleotideencoding the binding molecule is expressed in the subject, where the oneor more molecules are associated with cell migration.28. The method of embodiment 27, wherein the AAV preparation may includeAAV vector particles, empty capsids, and host cell impurities, therebyproviding an AAV product substantially free of AAV empty capsids.29. A pharmaceutical composition comprising a population of the cells ofany one of embodiments 24-26.30. A method of causing or eliciting T cell response in a subject inneed thereof and/or treating a tumor of the subject, the methodcomprising administering an effective amount of the composition ofembodiment 29 to the subject.31. An isolated nucleic acid sequence encoding one or more moleculesthat are associated with cell migration.32. The isolated nucleic acid sequence, modified cell, method, orpharmaceutical composition of any one of embodiments 24-31, wherein theone or more molecules comprise at least one of DOCKS, CDC42, and LRCH1,a functional variant of the one or more molecules, or a functionalfragment of the one or more molecules.33. The isolated nucleic acid sequence, modified cell, method, orpharmaceutical composition of any one of embodiments 24-32, wherein theone or more molecules are or comprise CDC42, which is overexpressed inthe modified T cells.34. The isolated nucleic acid sequence, modified cell, method, orpharmaceutical composition of any one of embodiments 24-33, wherein theone or more molecules are or comprise LRCH1 of which expression isreduced in the modified T cells.35. The isolated nucleic acid sequence, modified cell, method, orpharmaceutical composition of any one of embodiments 24-34, wherein themodified cell comprises a recombinant polynucleotide encoding the SEQ IDNOS: 43 and/or 39.36. The modified cell of any one of the preceding embodiments, whereinthe modified cell comprises an antigen binding molecule, wherein achimeric antigen receptor (CAR) comprises the antigen binding molecule,and wherein the CAR comprises an antigen-binding domain, a transmembranedomain, and an intracellular signaling domain.37. The modified cell of embodiment 36, wherein the antigen-bindingdomain binds to a tumor antigen selected from the group consisting of:TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2,GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA,EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2,LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2(Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-Ireceptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1,sLe, GM3, TGSS, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248,TEM7R, CLDN6, GPRCSD, CXORF61, CD97, CD179a, ALK, Polysialic acid,PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2,TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1,ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2,Fos-related antigen 1, p53, p53 mutant, prostein, survivin andtelomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcomatranslocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17,PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS,SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerasereverse transcriptase, RU1, RU2, intestinal carboxyl esterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A,BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.38. The modified cell of embodiment 36 and 37, wherein the intracellularsignaling domain comprises a co-stimulatory signaling domain, or aprimary signaling domain and a co-stimulatory signaling domain, whereinthe co-stimulatory signaling domain comprises a functional signalingdomain of a protein selected from the group consisting of CD27, CD28,4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta,IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4,CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a,LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1,ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44,NKp30, NKp46, and NKG2D39. The modified cell of any one of embodiments 24-30 or 31-35, whereinthe modified cell comprises the antigen binding molecule, and a modifiedTCR comprises the antigen binding molecule.40. The modified cell of embodiment 39, wherein the TCR is derived fromspontaneously occurring tumor-specific T cells in patients.41. The modified cell of embodiment 40, wherein the TCR binds a tumorantigen.42. The modified cell of embodiment 41, wherein the tumor antigencomprises CEA, gp100, MART-1, p53, MAGE-A3, or NY-ESO-1.43. The modified cell of embodiment 41, wherein the TCR comprises TCRγand TCRδ chains, or TCRα and TCRβ chains, or a combination thereof.44. The modified cell of any one of the preceding embodiments, whereinthe cell is an immune effector cell. (e.g., a population of immuneeffector cells).45. The modified cell of embodiment 44, wherein the immune effector cellis a T cell or an NK cell.46. The modified cell of embodiment 45, wherein the immune effector cellis a T cell.47. The modified cell of embodiment 45, wherein the T cell is a CD4+ Tcell, a CD8+ T cell, or a combination thereof.48. The modified cell of any one of the preceding embodiments, whereinthe cell is a human cell.49. The modified cell of any one of the preceding embodiments, whereinthe enhanced expression and/or function of the one or more molecules areimplemented by introducing a nucleic acid sequence encoding the one ormore molecules and/or the binding molecule, which is present in themodified cell in a recombinant DNA construct, in an mRNA, or in a viralvector.50. The modified cell of embodiment 49, wherein the nucleic acidsequence is an mRNA, which is not integrated into the genome of themodified cell.51. The modified cell of embodiment 49, wherein the nucleic acidsequence is associated with an oxygen-sensitive polypeptide domain.52. The modified cell of embodiment 51, wherein the oxygen-sensitivepolypeptide domain comprises HIF VHL binding domain.53. The modified cell of embodiment 49, wherein the nucleic acidsequence is regulated by a promoter comprising a binding site for atranscription modulator that modulates the expression of the CDC42 inthe cell.54. The modified cell of embodiment 53, wherein the transcriptionmodulator is or includes Hif1a, NFAT, FOXP3, and/or NFkB.55. A pharmaceutical composition comprising a population of the cells ofany one of embodiments 1-54.56. A method of causing or eliciting T cell response in a subject inneed thereof and/or treating a tumor of the subject, the methodcomprising administering an effective amount of the composition of claim55 to the subject.

Examples Cells Expressing Chimeric Receptors

Lentiviral vectors that encode individual CAR molecules were generatedand transfected with T cells, which are elaborated below. Techniquesrelated to cell cultures, construction of cytotoxic T-lymphocyte assaymay be found in “Control of large, established tumor xenografts withgenetically retargeted human T cells containing CD28 and CD137 domains,”PNAS, Mar. 3, 2009, vol. 106 no. 9, 3360-3365 and “Chimeric ReceptorsContaining CD137 Signal Transduction Domains Mediate Enhanced Survivalof T Cells and Increased Antileukemic Efficacy In Vivo,” MolecularTherapy, August 2009, vol. 17 no. 8, 1453-1464, which are incorporatedherein by reference in its entirety. Corresponding sequences can befound in Table 1.

TABLE 1 Sequences and Identifiers SEQ ID Identifier SEQ ID Identifier 1SP 22 LRCH1 2 Linker 23 Target for ZFN 3 4-1BB 24 Target for Talen 4CD3-zeta 25 Target for Crisper/Cas9 5 WT CD3-zeta-aa 26 ZFN Left F1 6Hinge & TM domain 27 ZFN Left F2 7 scFv CD19 28 ZFN Left F3 8 scFvHumanized CD19 29 ZFN Left F4 9 scFv FZD10 30 ZFN Right F1 10 scFv TSHR31 ZFN Right F2 11 scFv PRLR 32 ZFN Right F3 12 scFv Muc 17 33 ZFN RightF4 13 scFv GUCY2C 34 Left arm TALEN 14 scFv CD207 35 Left arm TALEN 15Prolactin (ligand) 36 Spacer 16 scFv CD3 37 Sh-RNA-SCREMBLE: 17 scFv CD438 Lrch1-shRNA1: 18 scFv CD4 39 scFv GUCY2C: 5F9 Nucleotide Sequence 19scFv CD5 40 P2A 20 ScFv MUC1-5e5 41 CD8hinge 21 ScFv MUC1-Panko 42 CDC42Nucleotide 45 5F9-CD8hinge-41bb-CD3Z: aa 43 CDC42 aa 465F9-CD8hinge-41bb-CD3Z-P2A-CDC42: Nucleotide 44 5F9-CD8hinge-41bb-CD3Z:Nucleotide 47 5F9-CD8hinge-41bb-CD3Z-P2A-CDC42: Aa

CAR T Cells Overexpressing CDC42

PBMCs were obtained from volunteers, and T cells were selected on Day 1.The T cells were introduced with a vector containing GUCY2C CAR and avector containing GUCY2C CAR-CDC42, respectively (See Table 2). Theinfected T cells were cultured until Day 7. On Day 7, 2×10⁵ T84 cellswere plated onto 24-well plates. On Day 8, 8×10⁵ GUCY2C CAR T cells, andGUCY2C CAR-CDC42 T cells were removed and added to T84-coated 24-wellplates. After coculturing for 24 hours, 3×10⁵ cells were removed todetermine the phenotype, and the supernatant was removed to determinethe release of GRAM B and IFN-γ. The phenotype was determined using aflow cytometry assay. FIG. 2 shows cytometry assay results indicatingthat CDC42 sequence does not affect the expression of CAR on T cells.FIG. 3 shows cytometry assay results indicating that CDC42 sequence doesnot affect the activation of CAR T cells. FIG. 4 shows CDC42 expressionenhances the release of GRAM B and IFN-γ after CAR activation. qPCR wasused to verify the efficiency of overexpressing CDC42. FIG. 5 shows thatCDC42 was successfully expressed in CAR T cells. On Day 7, 2×10⁶ GUCY2CCAR T cells, and GUCY2C CAR-CDC42 cells were used to verify theefficiency of overexpressing CDC42, respectively. RNA was extractedusing TRAzol, reverse transcription was performed using this RNA as atemplate, and qPCR was performed using reverse transcript as a templateto measure the overexpression of CDC42.

TABLE 2 ID Construct structure GUCY2C 5F9-CD8hinge-41BB-CD3Z GUC-CDC425F9-CD8hinge-41BB-CD3Z-P2A-CDC42 GUCY2C 5F9-CD8hinge-41BB-CD3Z GUC-SCRU6-shRNA-Scremble-5F9-CD8hinge-41BB-CD3Z ShRNA1U6-shRNA-1-5F9-CD8hinge-41BB-CD3Z

Overexpression CDC42 Enhance CAR T Cells' Migration Ability

The effect of overexpressing CDC42 on cell migration ability wasverified by Transwell assay. On Day 9, 2×10⁵ GUCY2C CAR T cells andGUCY2C CAR-CDC42 cells were placed in separate Transwell upper chamber.600 ul of X-VIVO containing 50 ng/ml CCL5 was placed in each of theTranswell lower chamber. Flow cytometry assay was performed after 4hours to measure the number of CAR+ cells that migrated in theunderlying medium. FIG. 6 shows that the migration ability of GUCY2C CART cells expressing CDC42 was significantly stronger than that of thecontrol group.

CAR T Cells Overexpressing LRCH1 and Enhanced Cytokine Releases

PBMCs were obtained from volunteers, and T cells were selected on Day 1.The T cells were introduced with a vector containing GUC-SCR and avector containing ShRNA1, respectively (See Table 2). The infected Tcells were cultured until Day 7. On Day 7, 2×10⁵ T84 cells were platedonto 24-well plates. On Day 8, 8×10⁵ GUC-SCR CAR T cells and ShRNA1 CART cells were removed and added to T84-coated 24-well plates. Aftercoculturing for 24 hours, 3×10⁵ cells were removed to determine thephenotype, and the supernatant was removed to determine the release ofGRAM B and IFN-γ. The phenotype was determined using flow cytometryassay. FIG. 7 shows cytometry assay results indicating that ShRNA1sequence does not affect the expression of CAR on T cells. FIG. 7 alsoshows cytometry assay results indicating that ShRNA1 sequence does notaffect the activation of CAR T cells. FIG. 8 shows ShRNA1 expressionenhances the release of GRAM B and IFN-γ after CAR activation. qPCR wasused to verify the efficiency of overexpressing CDC42.

All publications, patents and patent applications cited in thisspecification are incorporated herein by reference in their entiretiesas if each individual publication, patent or patent application werespecifically and individually indicated to be incorporated by reference.While the foregoing has been described in terms of various embodiments,the skilled artisan will appreciate that various modifications,substitutions, omissions, and changes may be made without departing fromthe spirit thereof.

1. A modified T cell comprising an antigen binding molecule, whereinexpression and/or function of CDC42 in the modified T cell has beenenhanced.
 2. The modified T cell of claim 1, where the modified T cellhas an increased level of a cytokine release in response to an antigenthat the antigen binding molecule binds as compared to a corresponding Tcell that does not overexpress CDC42.
 3. The modified T cell of claim 2,wherein the cytokine release comprises a cytokine release of IFNγ. 4.The modified T cell of claim 1, where the modified T cell has anincreased level of a protease release in response to an antigen that theantigen binding molecule binds as compared to a corresponding T cellthat does not overexpress CDC42.
 5. The modified T cell of claim 3,wherein the cytokine release comprises a protease release of GRAM B. 6.The modified T cell of claim 1, where the modified cell has an enhancedmigration capability in response to a chemokine as compared to acorresponding T cell that does not overexpress CDC42.
 7. The modified Tcell of claim 6, wherein the chemokine is CCL5.
 8. The modified T cellof claim 1, wherein the modified T cell comprises a polynucleotideencoding SEQ ID NO: 43 and the antigen binding molecule.
 9. The modifiedT cell of claim 1, wherein the modified T cell comprises a chimericantigen receptor (CAR) comprising the antigen binding domain, atransmembrane domain, and an intracellular signaling domain.
 10. Themodified T cell of claim 9, wherein the antigen binding domain binds atumor antigen.
 11. The modified T cell of claim 9, wherein the tumorantigen is selected from the group consisting of: TSHR, CD19, CD123,CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag,PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT,IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24,PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1,EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2,gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGSS, HMWMAA,o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRCSD,CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1,UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1,LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8,MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints,ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor,Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK,AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2,intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1,FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, andIGLL1.
 12. The modified T cell of claim 11, wherein the intracellularsignaling domain comprises a co-stimulatory signaling domain, or aprimary signaling domain and a co-stimulatory signaling domain.
 13. Themodified T cell of claim 12, wherein the co-stimulatory signaling domaincomprises a functional signaling domain of a protein selected from thegroup consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1,GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4,CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D.
 14. The modified T cellof claim 1, wherein the modified T cell comprises a TCR or modified TCRcomprising the antigen binding molecule.
 15. The modified T cell ofclaim 14, wherein the TCR is derived from spontaneously occurringtumor-specific T cells in patients.
 16. The modified T cell of claim 15,wherein the TCR binds a tumor antigen.
 17. The modified T cell of claim1, wherein the modified T cell comprises a polynucleotide encoding SEQID NO: 43, and the polynucleotide is present in the modified cell in arecombinant DNA construct, in an mRNA, or in a viral vector.
 18. Themodified T cell of claim 17, wherein the polynucleotide is regulated bya promoter comprising a binding site for a transcription modulator thatmodulates the expression of the CDC42 in the modified T cell.
 19. Themodified T cell of claim 18, wherein the transcription modulatorcomprises Hif1a, NFAT, FOXP3, and/or NFkB.
 20. A pharmaceuticalcomposition comprising the modified T cells of claim
 1. 21. A method ofcausing or eliciting T cell response in a subject in need thereof and/ortreating a tumor of the subject, the method comprising administering aneffective amount of the composition of claim 18 to the subject.
 22. Anisolated nucleic acid comprising a polynucleotide encoding SEQ ID NO:43.